Our long-term goal is to elucidate the molecular mechanisms by which thyroid stem cells differentiate into definitive thyrocytes. We specifically focus on thyroid stimulating hormone (TSH), as it is the major hormone in the control of thyroid growth and function. To examine whether TSH directly regulates thyroid development, we generated a TSHR null mouse by replacing exon 1 of the mTSHR gene with green fluorescent protein. We found that TSHR'''mice were severely hypothyroid and died at 4 weeks. Heterozygous mice were unaffected. Intense green fluorescence was detected in thyroid follicles of both TSHR +/ - and TSHR-/- mice. Of note is that follicles in TSHR-/- mice were poorly developed with fewer follicular cells, implying that the follicular growth and differentiation were dependent on TSH. To access early embryonic stages of development, we obtained the TSHR +/ - ES cell line. In addition to providing functional characterization of the TSHR in the thyroid in vivo, these ES cells have enabled us to track thyrocyte induction and its specification to the thyroid cell lineage in the ES cell differentiation model. Two specific aims are proposed. Specific aim 1 will focus on the induction and specification of thyroid lineage during ES cell differentiation. We will investigate when are TSHR and TSH expressed during in vitro differentiation of TSHR +/ - and TSHR -/- ES cells. We will also examine the temporal and spatial expression of TSHR relative to differentiation markers indicative of thyroid lineages in TSHR +/- and TSHR -/- ES cells. More importantly, we will characterize TSH/TSHR signaling in thyrocyte development and investigate whether insulin and insulin growth factor-1 can up-regulate thyroglobulin, a characteristic of mature thyrocytes. Specific Aim 2 will investigate the in vivo thyrocyte potential of TSHR-positive populations using actual murine embryos or ES cell differentiation model. First, we will further characterize the thyroid phenotypes of TSHR +/ - and TSHR -/- mouse embryos during postimplantation development. We will next investigate whether TSHR-positive cells from embryonic tissues have thyrocyte potential. Finally, we will determine whether TSHR-positive cells from ES cells can differentiate into functional thyrocytes in SCID mice. The outcome of these experiments will provide a clearer understanding of the characteristic of embryonic thyroid cells and of how these cells relate to definitive thyrocyte lineage. These studies may have important implications not only for our understanding of normal thyroid development but may provide insights into mechanisms underlying human diseases.